The proposed research will study the general mechanism of action of aldolase-type enzymes through the use of O18-labeled substrates. The objective of this work is to obtain data which will show unequivocally which of the proposed mechanisms operate in each of a variety of isolated enzymes. This will be accomplished by determining the fate of the O18 isotope incorporated into the keto oxygen atom of the various substrates. Direct analysis for both the abundance and position of the isotope in the product will be accomplished by mass spectrometry. Two experimental approaches will be taken. The first involves the rapid conversion of dihydroxyacetone phosphate (DHAP) to L-alpha- glycerophosphate by the action of L-alpha-glycerophosphate dehydrogenase. This is done to prevent loss of O18 due to the fast non- enzymatic exchange of the keto oxygen atom of DHAP. Preliminary experiments show that this approach will be successful. A Schiff base mechanism for aldolase would cause an obligatory loss of the O18 keto oxygen atom of the substrate to the medium, while a metal chelate mechanism would not. The second experimental approach involves the use of fructose diphosphate labeled with deuterium and O18 and is designed to measure the amount of non-obligatory enzyme catalyzed exchange of the keto oxygen of the substrate. Mass spectrometric analysis will be used to choose only those species of labeled molecules which have undergone dealdolization and condensation where DHAP has not been released from the enzyme. Two groups of enzymes will be studied. The first is fructose 1,6- diphosphate aldolase isolated from various sources, i.e., rabbit muscle, yeast, E. coli, M. aerogenes, and spinach. The second group is aldolases catalyzing reaction with different substrates but which produce DHAP from their respective substrates, i.e., fuculose 1- phosphate aldolase, rhamulose 1-phosphate aldolase and phosphoketotetrose aldolase.